Objective To construct the eukaryotic expression vector containing short hairpin RNA (shRNA) targeting the inhibition of Smad3 gene segment in keloid fibroblasts (KFBs) and to investigate the effects on the expression of Smad7 in KFBs. Methods A couple of the most effective siRNA screened from former experiments were used to recombine the plasmids of Smad3 shRNA, and then Smad3 shRNA was transfected into KFBs by LyoVec TM. The expressions of Smad3 and Smad7 at different time points (0～9 d) were detected by RT-PCR and Western blotting. Results ① The recombinant Smad3 shRNA vectors were identified by RT-PCR and Western blotting. ② The expressions of mRNA and protein of Smad3 in KFBs decreased significantly in a time-dependent manner after transfection of Smad3 shRNA and reached the lowest point at 72 hours in the experimental group. Optical density analysis revealed a significant difference between the experimental group and the control group (P

Objective To study the role of src-suppressed C kinase substrate (SSeCKS) in the secretion of tumor necrosis factor (TNF-α) in rat pulmonary micro-vascular endothelial cells (PMVEC) induced by lipopolysaccharide (LPS).Methods Wistar rat PMVEC cultured in vitro were randomly (random number) divided into several groups (n =4) as per exposure to given dosage of LPS for different lengths of time and to different dosages of LPS for given length of time.After PMVEC exposed to 10 mg/L LPS for 1 hour (h),3 h,6 h,12 h and 24 h or 0.1 mg/L,1 mg/L and 10 mg/L LPS for 24 h,the levels of TNF-αin the supernatant of culture medium were examined by the method of enzyme linked immunosorbent assay (ELISA).Another PMVEC was pre-treated by protein kinase C (PKC) inhibitor bis-indolylmaleimide (BIM) for 0.5 h or had the transfection of SSeCKS-specific small interfering RNA (siRNA) for 48 h before 10 mg/L LPS challenge for 24 h,and subsequently the supernatant was also examined by ELISA.One-way analysis of variance (ANOVA) was employed for statistical analysis by SPSS version 10.0 to compare values among all groups.A significant difference was presumed as a probability value ＜ 0.05.Results After PMVEC incubated with 0.1 mg/L,1 mg/L and 10 mg/L LPS for 24 hours,the levels of TNF-αsecreted were (253.70 ± 23.55),(327.88 ± 37.25),(403.20 ± 36.22),respectively,which were higher than that in un-stimulated PMVEC (82.28 ± 22.56,all P =0.000).After 10 mg/L LPS challenge for one hour,the level of TNF-αin the supernatant of PMVEC raised substantially (170.11 ±49.22),peaked at the time of 6 h (404.82 ± 13.78),then persisted at a higher level until 24 h (395.67 ± 36.23) than that in un-stimulated PMVEC (84.60 ± 23.61,P =0.001,0.000,0.000,respectively).After PMVEC pre-incubated with BIM,the level of LPS-induced TNF-αdecreased obviously (200.44 ± 27.39 vs.402.28 ± 31.07,P =0.000).Compared with LPS challenged PMVEC (407.28 ± 32.64),depletion of endogenous SSeCKS in PMVEC after inhibited by SSeCKS-siRNA significantly attenuated increase in the level of LPS-induced TNF-α (195.20 ± 13.28,P =0.000).Conclusions Down-activation of SSeCKS and PKC can inhibit the secretion of TNF-αin PMVEC induced by LPS,relieving the inflammatory response of PMVEC.

Myoblast line C_2C_ 12 cells were co-cultured with various concentrations of glucagon-like peptide-1 (GLP-1). Being induced by GLP-1, myoblast line C_2C_ 12 cells have the potential to differentiate into cells which are able to secrete insulin-like substance.

Objective To explore the value of contrast-enhanced ultrasound(CEUS) in diagnosis of placental accrete,placental increta and placental percreta. Methods Twenty suspected patients of placental accrete, placental increta and placental percreta were examined with CEUS after routine ultrasound examination. Sono Vue was injected intravenously as bolus and a real time CEUS was performed to observe the characteristics. Results Comparison of 15 CEUS cases with uterine curettage, 8 cases had a clear relationship between the residual disease of the uterine cavity and uterine wall,7 cases had a thin thickness (2-4 mm) between the residual disease of the uterine cavity and uterine wall. Five of the 20 cases performed cesarean cesion,3 cases of the uterine serosa of placenta affixing part were very thin,incomplete and rough, demonstrating placenta increta and placenta percreta, 1 case of uterine rupture showed non-invasiveness between uterine wall and uterine serosa,and 1 case of placenta increta pathologically proven, initially suspected gestational trophoblastic disease by the CEUS. Conclusions CEUS provides an important diagnostic message for placental accrete, placental increta and placental percreta by showing the different characteristics of the perfusion image.

This study examined the effect of long-term administration of low-dose FTY720 on survival of murine cardiac allograft and the possible mechanism. Murine models of abdominal heterotopic heart transplantation were established. Low-dose FTY720 (0.3 mg/kg) was administrated to the animals 4 days before the transplantation of cardiac allografts until the occurrence of rejection or the observation terminals. The animals without FTY720 treatment and those with syngeneic cardiac grafts transplanted served as controls. The mean survival time (MST) of grafts, and T lymphocyte subsets in grafts, peripheral blood and lymphoid organs were measured by histopathological examination or flow cytometry, and compared among groups. The results showed that the MST of allografts in FTY720-treated mice was more than 40 days, significantly longer than that in the untreated group (MST=8 days, P<0.01). After the long-term administration of FTY720, the proportion of CD4(+) and CD8(+) lymphocytes in peripheral blood was diminished significantly, but the proportion of CD4(+) lymphocytes was increased in mesenteric lymph nodes (MLNs) and spleen. Immunofluorescence staining revealed that the infiltration of CD4(+) and CD8(+) lymphocytes in allografts was significantly inhibited after long-term administration of low-dose FTY720. It was concluded that low-dose long-term administration of FTY720 could promote T lymphocytes in lymphatic organs and decrease their infiltration in allografts, resulting in the inhibition of rejection and the long-term survival of allografts.

Objective To investigate the accuracy of low-dose dual-source computed tomography (DSCT) coronary angiography in the step-and-shoot (SAS) mode for the diagnosis of coronary artery stenosis in comparison with conventional coronary angingraphy (CCA).Methods Prospective multiple-center study, 46 patients[mean age(58±9) years;bedy mass index(BMI) (25±3) kg/m2]underwent both DSCT in the SAS mode and CCA within 14 days.The inclusion criteria for contrast-enhanced CT: (1) heart rate less than 65 times/rain (bpm).(2) regular sinus rhythm, heart rate fluctuations within the range of 6 bpm. (3) holding breath well, breath-hold time is about 12-15 s.The exclusion criteria:(1) allergy to iodinecontaining contrast medium, nephropathy (serum creatinine level 120 μmol/L), heart failure and serious arrhythmias.(2) patients with coronary stents or bypass grafts.(3) heart rate can not be controlled very well (4)the patient could not take nitroglycerin.(5)BMI 30 kg/m2.(6) other heart disease: carcliomyopathy, valvular disease etc.Sensitivity, specificity, negative (NPV) and positive predictive value (PPV) were determined with CCA as standard of reference.The Kappa value between the two modalities and the two observers was calculated.Radiation dose values were measured.Results Mean heart rate during scanning was (61±6)bpm.99.19% (614/619) coronary segments were depicted with a diagnostic image quality. The vessel-based sensitivity, specificity, PPV, and NPV for the diagnosis of coronary artery stenosis were 96.2% (75/78), 88.2% (60/68), 90.4% (75/83), and 95.2% (60/63), respectively.The Kappa value between the two modalities was 0.848 (P=0.000).The mean effective dose of the SAS-CTCA was (2.95± 0.96) rosy(1.26-4.32 mSy).Conclusion In selected patients, DSCT coronary angiography in the SAS mode have good image quality, which allows for the accurate diagnosis of coronary stenosis at a low radiation dose.

ObjectiveTo study the effect of fluorine on proliferation of osteoblast through extra cellular signal-regulated protein kinase(ERK) signaling pathway.MethodsMouse osteoblasts(MC3T3-E1) were cultured in vitro with different concentrations of fluoride for 24 and 48 h (the concentrations of Fˉ were 0,200,400,600,1000,2000,4000,8000,10 000 μmol/L,respectively).The optimum concentration for promotion of cell proliferation was determined by methylthiophene tetrazolium(MTT) assay.According to the optimum concentration,the cells were randomly divided into three groups:control group (0 μmol/L Fˉ); fluorine group (400 μmol/L Fˉ); fluorine and MAPK inhibitor PD98059 group(400 μ mol/L Fˉ + 10 μ mmol/L PD98059).Cell cycle was detected by flow cytometry after 48 h culture.The expression of P-ERK protein was determined by Western blotting and immunofluorescence.ResultsThe optimum concentration of fluorine for proliferation of osteoblasts was 400 μ mol/L.Compared with the control group[(76.12 ± 10.08)％,(2.06 ± 0.31)％],the number of cells in G0/G1 phase[(63.04 ± 8.12)％] reduced and the number of cells in S phase[(9.13 ± 2.08)％] increased in fluorine group (all P ＜ 0.05) ; but the number of cells in G0/G1 phase [(92.11 ± 9.01 ) ％] in fluorine and mitogen-activated protein kinases (MAPK) inhibitor PD98059 group was significantly increased(P ＜ 0.05 ).Western blotting results showed that:compared with the control group[(100.00 ± 0.00)％],the expression of P-ERK protein in fluorine group[(131.24 ± 13.88)％] was significantly higher(P ＜ 0.05 ),but the expression of P-ERK protein in fluorine and MAPK inhibitor PD98059 group [(91.33 ± 9.68 )％] was not significantly changed(P ＞ 0.05).The results of immunofluorescence were similar to that of Western blotting.ConclusionsFluorine at the concentration of 400 μmol/L can promote the proliferation of osteoblasts.ERK signaling pathway has played a key role in the proliferation of osteoblasts.

Objective To observe the expressions of OPN, COX-2 and CyclinD1 in breast infiltrating carcinoma and evaluate their relationships with clinic pathological features. Methods Expression of the above three indexes were detected from 70 breast cancinoma patients by immunohistochemistry. The relationships among them and clinicopathological features were analyzed. Results The positive expression rates of OPN were 78.8% in cases (≤45 years old) and 73.0% in cases (> 45 years old); the positive expression rates were 79.3%(tumor diameter ≤ 3 cm) and 73.2% (tumor diameter > 3 cm); the positive expression rates were 77.8%, 73.8% and 78.9% in cases ofⅠgrade, Ⅱgrade and Ⅲ respectively, the positive rates had no statistical significances(P > 0.05). The expression rates of OPN in cases of breast infiltrating carcinoma without and with axillary node metastasis were 62.5% and 93.3%, in cases at stage Ⅰ~Ⅱ and Ⅲ ~Ⅳ were, 68.0% and 95.0% respectively, the positive rates had statistical significances(P < 0.05). The expression of OPN was negatively correlated with ER and PR while positively correlated with CerbB2, COX-2 and CyclinD1. Conclusions OPN plays an important role in the invasion and metastasis of breast carcinoma coordinated with COX-2 and CyclinD1.

Objective Analyse the change of left ventricular (LV) longitudinal and radial strain in patients with aortic regurgitation (AR) and discuss the relationship between the 2D strain parameter and the filling and ejection of LV. Methods Thirty healthy controls and 45 patients with AR (24 patients with moderate AR and 21 with severe AR) were enrolled in this study, LV systolic global peak radial strain(GRS), systolic global peak longitudinal strain(GLS) and systolic peak longitudinal strain(S), systolic peak longitudinal strain rate(SRs), early diastolic peak longitudinal strain rate(SRe) of every segment were measured or calculated using 2D-STE, early and late diastolic transmitral flow velocity (E, A) were recorded by pulsed Doppler echocardiography and early diastolic mitral annular velocity (Ea) were assessed by tissue Doppler imaging,the E/A and E/Ea ratio were calculated. Discuss the relationship of GLS and LV ejection fraction (LVEF), GLS and E/Ea using the Pearson correlation analysis. Results The GLS were (-20.09±1.47)%, (-18.68±1.52)%, (-12.56±3.25)%and the GRS were (46.71±7.65)%, (43.01±5.95)%, (28.52±6.13)% in control group, patients with moderate and severe AR (MAR group and SAR group) respectively. There were significant differences among the groups (F =82.08,47.69, both P < 0.01) as following:SAR group with control group and MAR group [ q=17.56,13.60 (GLS), q=13.44, 10.20 (GRS), all P<0.01),MAR group and control group [ q=3.42 (GLS), P<0.01]. The SRs of the apical segment were (-1.24±0.22)s-1, (-1.19±0.25)s-1, (-1.04±0.28)s-1 in control group,MAR group and SAR group respectively. There were significant differences among the groups (F=4.47, P < 0.05) as following:SAR group with control group and MAR group ( q=4.02,3.28, both P<0.01). The S, SRe of apical segment and the S,SRs,SRe of basal and midventricular in MAR group were all lower than the control group ( q=4.42, 5.01, 3.48, 3.24, 4.78, 4.12, 3.61, 6.72, all P < 0.01). Pearson correlation analysis revealed the GLS had a relationship with LVEF and E/Ea ( r=-0.73, 0.64, both P<0.01). Conclusion The reduced longitudinal strain and strain rate could detect LV dysfunction in patients with AR in early stage and the GLS had the ability to reflect the diastolic filling and systolic ejecting of the LV.

AIM:To investigate the autophagy induced by sepsis and acute kidney injury , and the regulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in this process.METHODS: The rats were subjected to cecal ligation and puncture ( CLP) or sham operation .Histopathologic changes of the renal tissues were examined by HE staining .Blood urea nitrogen ( BUN) and serum creatinine ( SCr) were measured by chemical colorime-try.The protein expression of microtubule-associated protein light chain 3 I/II (LC3 I/II), beclin-1 and p-Akt at different time points after CLP was detected by Western blotting .In vitro, human proximal tubular epithelial cell line HK-2 were treated with LPS to induce autophagy .The protein expression of LC 3 I/II and p-Akt in the HK-2 cells after LPS treatment at different time points and different concentrations was detected by Western blotting .These molecules in HK-2 cells and apoptosis of HK-2 cells treated with LPS plus PI3K inhibitor or Akt inhibitor were also detected .RESULTS: Compared with sham group , the severe changes of renal histopathological injuries in CLP groups were observed , the levels of BUN and SCr in CLP groups were significantly increased .LC3 I/II, beclin-1 and phosphorylation of Akt gradually increased after CLP.After treatment with LPS, the expression of p-Akt (308) in the HK-2 cells gradually increased in a dose-and time-dependent fashion.The expression of beclin-1 and p-Akt (472) reached a peak at 8 h or 10 mg/L LPS treatment.Treat-ment with PI3K or Akt inhibitor down-regulated the expression of LC3 and promoted the apoptosis of HK-2 cells.CON-CLUSION:Autophagy in the kidney is induced by sepsis and acute kidney injury .PI3/Akt signaling pathway may be in-volved in this process .